Method for nonspecific cellular immune stimulation

ABSTRACT

A non-specific host defense cell augmentation technique for enhanced microorganism killing utilizes any phagocytosable, biocompatible particle to prime macrophages for enhanced oxidative response and bacterial killing. Patients can have the benefits of primed macrophages in one to four days, and experiments have demonstrated over a 100-fold increase in oxidative potential within this time period. The oxidative response and killing potential is non-immunospecific, meaning not one organism, not a vaccine, and broadly applicable simultaneously to bacteria and viruses as well as tumor cells. The effects have been demonstrated to have a seven day duration indicating non-tissue toxic residual effects and potential for repeated use at monthly intervals.

DESCRIPTION

1. Phagocyte-a cell that engulfs bacteria and other foreign particles byphagocytosis.

2. Macrophage-a cell derived from the reticuloendothelial system thatfunctions in phagocytosis. Macrophages are phagocytes.

3. Activate-transforming a cell from a resting state to one where itactively performs its biological function. For example, a macrophage orphagocyte is activated when it encounters a foreign object. Uponencountering the foreign object, the macrophage releases a respiratoryburst of oxidizing chemicals to kill or otherwise destroy the object.

4. Elicit-to evoke a response from a cell. For example, foreign objectsmight be provided to macrophages to elicit the respiratory burstactivity.

5. Priming-converting a cell from one state to another, whereby itsprimed state is more active to a biological substance than if the cellhad not been primed. In this patent, the difference between priming amacrophage, as opposed to activating a macrophage or eliciting aresponse, is very important.

6. Cytokine-a group of substances formed by an animal in response toinfection. Cytokines are similar to hormones in their function, wherebythey are produced in one cell and stimulate a response in another cell.Cytokines includes such substances as interferon, interleukin, and tumornecrosis factor.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is generally related to priming macrophages for enhancedkilling potential. More particularly, the invention includesadministering a priming factor to a patient so that the macrophages inthe patient will be primed for enhanced killing activity a certainnumber of days after the priming factor was administered.

2. Description of the Prior Art

It is now well understood that phagocytes, such as alveolar macrophagesand the like, play an important role in controlling microbialinfections. Baboir, New Eng. J. Med., 298:659-68 (1978), has explainedthat upon encountering a foreign material, such as an invading bacterialcell, phagocytes produce a respiratory burst wherein highly oxidativespecies, such as superoxide anion (O₂ ⁻), singlet oxygen (O₂), andhydrogen peroxide (H₂ O₂), are produced. The purpose of the respiratoryburst is to provide a battery of oxidizing agents that can be used bythe phagocyte for the destruction of invading micro-organisms and otherforeign material. Many agents, both particulate and soluble, are able toactivate the respiratory burst. Particulate activating agents includeopsonized bacteria, zymosan (a preparation of yeast cell walls), andlatex spheres. Among the soluble activating agents are phorbol myristateacetate, a complex plant product; a variety of ionophores; thecomplement C5a; and fluoride ion. Activation may not requirephagocytosis; rather, simply contact of the foreign stimulant with thephagocyte surface may be enough to activate the phagocyte to produce therespiratory burst. The oxygen-dependent cytotoxic mechanisms ofphagocytes are discussed at length in Klebanoff, Adv. Host Def. Mech.,(Vol. 1, eds. J. Gallin and A. Fauci, Raven Press, New York 1982pp.111-163).

Baboir also explains that the respiratory burst activity can be detectedby monitoring the chemiluminescence phenomena wherein light emissionaccompanies activation of the phagocyte. The light emission stems fromthe oxidative species produced by the phagocyte. For example, singletoxygen is an electronically excited state of oxygen that can revertspontaneously to atmospheric oxygen, and this reversion is accompaniedby a pulse of light. However, it is now generally believed thatsuperoxide anion is responsible for the chemiluminescent response.

Many researcher groups have used chemiluminescence to study macrophageactivity. For example, Donaldson et al., Br. J. exp. Path., 65:81-90(1984), used chemiluminescence measurements to show that macrophagestreated with chrysotile asbestos and Cornyebacterium parvum elicitedgreater levels of reactive oxygen species than saline treatedmacrophages. In addition, Donaldson showed that peritoneal exudate cellsharvested from CF_(I) mice five days after injection with chrysotileasbestos or C. parvum had a approximately a two to three fold increasein measured chemiluminescence. Donaldson et al. suggest that theasbestos-activated macrophages are primed to produce increased amountsof reactive oxygen species which could be triggered by a number ofinhalable particles (e.g., bacteria, yeast, pollen, and asbestositself), and that an excess of these reactive oxygen species in thealveolar spaces leads to epithelial damage and ultimately to fibrosis.Other examples where chemiluminescent response measurements were usedinclude: Chida et al., Infect. Immun., 55:1476-1483 (1987), reports on astudy where infant and mature rabbits were vaccinated with the heatkilled Bacillus Calmette Guerin (BCG) strain of Myobacterium bovis andshows that the alveolar macrophages (AM) of infant rabbits were poorresponders to phorbol myristate acetate (PMA)-induced chemiluminescentresponses compared to AM from older rabbits which were vaccinated withBCG, thus illustrating a deficiency in the AM of neonatal and infantanimals that may account for their increased susceptibility to pulmonaryinfections; Hayakawa et al., J. Leuk. Biol., 45:231- 238 (1989), reportson a study where a chemiluminescent assay was used to show that AM fromBCG vaccinated rabbits (3 weeks after i.v. injection), when cultured invitro with various serum preparations, could result in significantchanges in the chemiluminescent (CL) response; Myrvik et al., J. Invest.Surg., 2:381-389 (1989), reports on a study where extracellular slimefrom Staphyloccocus epidermis was found to affect the CL response onPMA-induced rabbit AM; Umehara et al., Cell. Immun., 119:67-72 (1989),reports on a study where CL responses were used to show L-Fucose blocksmigration inhibition factor (MIF)/macrophage activation factor (MAF)priming of rabbit AM (PMA-induced oxidative response used); Giridhar etal., J. Leuk. Biol., 49:442-448 (1991), reports on a study where CLresponses were used to show priming of rabbit AM by herpes simplex virustype 2 infection.

There has been much effort made in finding materials which can provideprotection from infection. U.S. Pat. Nos. 4,707,471 and 4,795,745 toLarm et al. disclose that pretreatment with water soluble aminatedβ-1,3-D-glucans can stimulate the activity of macrophages such thatanimals are protected from virulent pneumococci. U.S. Pat. No. 5,045,320to Mescher discloses that immunization with a solid support having avariety of different ligands attached can elicit and augment T cellmediated responses U.S. Pat. No. 4,900,722 to Williams et al. disclosesa class of phosphorylated glucans useful in the treatment of infections.U.S. Pat. No. 5,078,996 to Conlon et al. discloses the use ofgranulocyte stimulating factor to activate macrophage tumoricidalactivity U.S. Pat. No. 3,119,741 discloses an acylated bacteriallipopolysaccharide useful as a non-specific immunological agent.

There is a need for a short-term, non-specific therapeutic whichprovides protection against a wide variety of bacterial and viralinfections. Such a therapeutic could ideally be used in anticipation ofevents which lead to infections such as surgery, biological warfare,natural disasters and the like. Up-regulation of the macrophageoxidative killing potential could be beneficial to such an end; however,the time duration for such priming would advantageously be limited so asto avoid cellular and matrix protein damage, fibrosis, and otherinjuries which would occur from the chronic production of reactiveoxygen species.

SUMMARY OF THE INVENTION

It is therefore an object of this invention to provide animmunomodulation technique for non-specific cellular immune stimulation.

It is another object of this invention to provide a method forup-regulating macrophages for a short duration by using phagocytosableparticulates to prime the macrophages for a short period of time.

It is yet another object of this invention to provide compositionssuitable for use in priming macrophages for enhanced killing potential.

According to the invention, macrophages can be primed for a markedlyenhanced oxidative response by injecting a patient with phagocytosableparticles a few days before the enhanced activity is required.Experiments suggest that the primed macrophages could have greater than100 times the activity potential than normal, non-primed macrophages.However, the priming is for a short duration and wears off to normalafter a week so that the treatment process does not pose long termhazards for enhanced in vivo reactive oxygen production.

In the experiments, adult rabbits were injected intravenously (i.v.)with phagocytosable (1-5 μm) particulate preparations such as zymosan,latex particles or heat-killed BCG. The preparations primed AM rapidlyin 1-4 days for greatly enhanced phorbol myristate acetate (PMA) oropsonized zymosan (Op-zym) elicited chemiluminescent (CL) responses. AMobtained from particle injected rabbits showed more than 100-fold higherlevels of CL responses than AM from normal rabbits Specifically, AM fromresident rabbits normally generate about 3,000 cpm when challenged withPMA, whereas AM from rabbits injected i.v. with 20 mg of zymosan threedays prior to harvesting AM, generated up to 900,000 cpm when challengedwith PMA. In contrast, the particles failed to prime normal AM in vitrofor enhanced CL responses. Furthermore, AM could not be primed in vivowith non-phagocytosable (-25 μm diameter) particles. The priming effectwas of short duration and declined 5 to 7 days after injection of theparticle preparations It was also observed that AM from normal rabbitscould be primed in vitro for enhanced CL responses by incubating AM for3 to 18 h with the lung lavage fluids obtained from particle-primedrabbits which suggests the presence of a macrophage priming factor(s) inthe lung lavage fluid.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, aspects and advantages will be betterunderstood from the following detailed description of a preferredembodiment of the invention with reference to the drawings, in which:

FIG. 1 is a graph showing in vitro priming of normal rabbit AM with BALextracted from rabbits injected with zymosan 1, 2, or 3 days prior toharvesting AM for enhanced PMA-elicited CL responses; and

FIG. 2 is a graph showing in vitro priming of normal rabbit AM withvarious concentrations of BAL extracted from zymosan injected rabbitsfor enhanced latex elicited CL responses.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

A number of experiments have been performed which demonstrate thatphagocytosable particles are effective for up-regulating macrophages forshort duration.

1. Materials and Methods Reagents

Tissue culture reagents were purchased from Curtin Matheson ScientificCompany, Inc. (Columbia, Md.). Polystyrene and PMMA latex particles werepurchased from Polysciences, Inc. (Warrington, Pa.). Other chemicalswere purchased from Sigma Chemical Company (St. Louis, Mo.) unlessotherwise specified.

Animals

New Zealand white SPF rabbits of either sex, 4-5 months old, werepurchased from Hazleton Research Products, Inc. (Denver, Pa.). Theanimals were housed for 3-4 days in our animal facility to allow theanimals to adjust to their new environment before being used.

Collection of Macrophages

Rabbits were sacrificed by pentobarbital given i.v. (75-85 mg/kg) orrabbits were anesthetized with Ketamine/rompon (40 mg/kg and 5-10 mg/kgi.m.) and then exsanguinated or given air embolism while underanesthesia. AM were harvested by lung lavage technique described inMyrik et al., J. Immunol. 86:128-132 (1961), which is hereinincorporated by reference, using 200 ml of cold saline. The harvestedcells were washed 3× by centrifugation (200× g, 10 min) in RPMI 1640medium (pH 7.2) containing penicillin (100 U/ml), streptomycin. (100μg/ml), and L-glutamine (2 mM), but without phenol red and serum. Thecells were resuspended in the same medium to obtain a cell density of3×10⁷ cells/ml.

Cell Viability

The viability of the AM was determined by trypan blue (0.25%) exclusionstaining.

Protocol for In Vivo Priming of Rabbit AM

To prime AM in vivo, adult rabbits were injected i.v. with 10 mg ofheat-killed BCG strain of Mycobacterium bovis suspended in 4 ml ofsaline or with 20 mg of zymosan in 2 ml of saline. The AM were harvested1 to 7 days after injection.

Chemiluminescence Assay

The CL response was assayed by a previously described procedure ofGirdhar et al., J. Leuk. Biol. 49:442-448 (1991), which is hereinincorporated by reference. The assay was done in diffused light usingdark-adapted 3.5 ml polypropylene scintillation vials. A typical assaymixture consisted of 3 ml Hanks' Balanced Salt Solution (HBSS) (pH 7.2)at 37° C., 0.1 ml cell suspension (3×10⁶ cells), and 30 μl, 0.5 μg/ml),latex (7.5 μl, 250 μg/ml), Polybead Polystyrene Microspheres, 2.5% solidlatex of 1.03 μm diameter, or opsonized zymosan (Op-zym), (30 μl; 100μg/ml) were added to elicit the CL response. The counts per minute (cpm)were recorded by scintillation spectrometry using a Beckman LS 100 Cscintillation counter.

Collection of Bronchoalveolar Lavage Fluids (BAL)

BAL were collected from control as well as particle-primed rabbits.Lungs removed from rabbits were lavaged using 100 ml of cold saline, andthe fluids were centrifuged at 300× g for 10 min to remove the cells andcell debris. The supernatant fluids were centrifuged at 60,000× g for 2h at 4° C., and the supernatant fluids were filtered (0.45 μm pore size)and used as crude BAL containing the putative macrophage priming factor(MPF).

In Vitro Priming of Normal Rabbit AM with BAL

Freshly-harvested AM from normal rabbits were incubated with variousconcentrations (10 to 100%) of BAL from primed animals for 3 to 18 h inRPMI 1640 medium at a cell density of 1×10₆ /ml in Teflon flasks. Afterincubation, the cells were washed 2× with RPMI 1640 medium and assayedfor a CL response with PMA or OP-zym as the eliciting agents. AMincubated with or without BAL from normal animals served as controls.

2. Experiments In Vivo Priming of AM in Adult Rabbits Following ZymosanAdministration for Enhanced Oxidative Responses

Adult rabbits were injected i.v. with 20 mg of zymosan particles in 2 mlof saline. On the days following injection, the animals were sacrificed,AM was harvested, and either PMA or Op-zym was used to elicitchemiluminescence from the harvested AM. Table 1 presents the measuredcpm*10⁻⁴ ±SEM for the two eliciting agents on the test days.

                  TABLE 1                                                         ______________________________________                                        Days          Oxidative Responses (CPM* 10.sup.-4)                            After         Eliciting Agents                                                Priming       PMA          Op-zym                                             ______________________________________                                        0 (control; no injection)                                                                   0.3 ± 0.5  7 ± 2.5                                        1             7.0 ± 1.1 17 ± 3.8                                        2             25 ± 5.2  45 ± 8.3                                        3             92 ± 10.1 >100                                               4             7 ± 2.5   12 ± 2.4                                        5             5 ± 0.3   21 ± 2.2                                        7             4 ± 0.4   25 ± 8.5                                        ______________________________________                                    

Table 1 shows a very dramatic short-term priming effect caused by theinjected particles. While the CPM values increased immediately afterinjection, unusually high levels of PMA- or Op-elicited oxidativeresponses was observed when particles were administered three days priorto harvest. AM harvested three days after zymosan injection generated aPMA-elicited CPM of more than 900,000 and Op-zym-elicited CPM weregreater than 1,000,000. In addition, an increase in the resting valuesfrom less than 500 CPM for normal AM to about 2,000 CPM for primed AMwas also observed (resting values being the CPM when no eliciting agentwas provided). The decline in the oxidative response observed from days4 to 7 demonstrates that the observed particle induced priming of themacrophages was for only a short duration.

Alternate modes of administration, such as intratracheal microdropletinstillation (e.g., aerosols and the like), yielded similar results.Adult rabbits were injected intratracheally (i.t.) with 20 mg of zymosanin 2 ml of saline four days prior to harvesting their AM. Controlrabbits received 2 ml of saline. The harvested AM were assayed for CLresponses with PMA or Op-zym as eliciting agents. Table 2 presents datashowing enhanced oxidative responses for AM obtained from rabbitsinjected i.t. with zymosan (data represent three separate experiments).

                  TABLE 2                                                         ______________________________________                                                      Oxidative responses (CPM* 10.sup.-4)                            Particle      Eliciting Agents                                                injected      PMA          Op-zym                                             ______________________________________                                        Saline (control)                                                                            0.2          5.1                                                Zymosan (20 mg)                                                                             14.6         >100                                               ______________________________________                                    

Table 2 shows that AM from rabbits injected i.t. with 20 mg of zymosan 4days prior to harvesting AM generated Op-zym elicited CL responses ofmore than 1,000,000 CPM.

Cell Analysis of Lavagates from Zymosan-Injected Animals and Evidencethat AM are Involved in the Priming Response

Adult rabbits were injected i.v. with 20 mg of zymosan in 2 ml ofsaline. Groups of rabbits were sacrificed and the cells (AM) wereharvested by lavage 1, 2, 3, or 4 days after zymosan injection. Theviability of the cells was determined by the trypan blue exclusion testand differential cell counts were determined by evaluatingcytocentrifuge slide preparations stained with Wright-Giemsa solution.Table 3 presents the differential numbers of AM, lymphocytes (Lym) andpolymorphonuclear leukocytes (PMN) from normal and zymosan injectedadult rabbits (results are ±SEM (n=4)).

                  TABLE 3                                                         ______________________________________                                        Days After iv                                                                            Differentiation of Cell Counts (%)                                 injection  AM          Lym      PMN                                           ______________________________________                                        Control    95.5 ± 22.0                                                                            5.0 ± 1.1                                                                           1.1 ± 0.2                                  1          72.2 ± 15.5                                                                            1.9 ± 0.7                                                                           24.7 ± 13.8                                2          78.5 ± 9.3                                                                             5.5 ± 2.6                                                                           16.2 ± 8.2                                 3          84.8 ± 0.8                                                                             9.0 ± 1.0                                                                           6.3 ± 2.2                                  4          95.4 ± 5.3                                                                             2.8 ± 3.1                                                                           1.9 ± 2.2                                  ______________________________________                                    

Table 3 shows that total neutrophils comprised about 25% of the totalrecovered on day 1, 16% on day 2, 6% on day 3 and 2% on day 4. In viewof the fact that AM from zymosan-injected rabbits exhibited about a 20to 200-fold increase in Op-zym or PMA elicited CL responses compared toAM from control rabbits (Table 1), and that the in vivo priming effectwas highest on day 3 when the AM population was 85% and the RMNpopulation was only about 6% of the cells harvested (Tables 1 and 3),the data establish AM was the predominant cell population involved inthe generation of the oxidative burst.

Comparative In Vivo Priming of AM From Adult Rabbits Following i.v.Injection of Zymosan, HK-BCG or Latex Particles

To study the comparative effects of other particles on primingmacrophages, adult rabbits were injected i.v. with 20 mg latex in 2 mlsaline, 20 mg zymosan in 2 ml of saline, or 10 mg heat killed (HK)-BCGin 4 ml of saline two days prior to harvesting AM. Oxidative responseswere then elicited by PMA (0.5 μg/ml), latex (100 μg/ml) or opsonizedzymosan (100 μg/ml). Table 4 presents data showing the in vivo primingof adult rabbit AM by zymosan, HK-BCG, and latex for enhanced oxidativeresponses elicited by PMA, latex, and op-zym.

                  TABLE 4                                                         ______________________________________                                                        Oxidative Responses (CPM* 10.sup.-4)                          Particles                                                                             Conc.   Eliciting Agents                                              injected                                                                              (mg)    PMA         Latex   Op-zym                                    ______________________________________                                        Control  0      0.25 ± 0.03                                                                            0.5 ± 0.2                                                                          7.5 ± 1.5                              Zymosan 20      6.2 ± 0.3                                                                              11.5 ± 2.2                                                                         46.0 ± 3.6                             HK-BCG  10      8.3 ± 0.4        >100                                      Latex   20      3.3                 14.9                                      ______________________________________                                    

Table 4 shows that HK-BCG and latex particles were also highly effectivein priming normal rabbit AM in vivo for markedly enhanced CL responses.Preliminary results with other particles have been similar. The resultsindicate that the in vivo priming of normal rabbit AM is non-specificwith respect to the types of particle preparations injected into therabbits as well as with respect to the eliciting agents. It ispreferable that the particles that are administered be biodegradablewithin a few days after their priming function is fulfilled.Specifically, particles should remain substantially intact for 1-4 daysto achieve the priming function presented in the above tables; however,after the fourth day, when the priming function has been found todiminish (Table 1), the particles would preferably be broken down bybodily functions so that the particles themselves would not present amedical challenge to the patient.

Failure of Non-Phagocytosable (˜25 μm) Latex Particles to Prime AM InVivo

In the above experiments, the particles employed were phagocytosable(e.g., 1-5 μm in diameter). To determine whether the size of theparticle plays an important role in priming the macrophages, adultrabbits were injected i.v. with 20 mg of non-phagocytosable latexparticles on the order of 25 μm in diameter. The latex beads weresuspended in 2 ml of saline. AM were harvested two days after injectionand assayed for PMA or Op-zym elicited CL responses. Table 5 shows thati.v. injection of adult rabbits in vivo with 20 mg of non-phagocytosablelatex particles approximately 25 μm in diameter did not result inpriming AM.

                  TABLE 5                                                         ______________________________________                                                     Oxidative Responses (CPM* 10.sup.-4)                             Source       Eliciting Agents                                                 of AM        PMA          Op-zym                                              ______________________________________                                        Control Rabbit                                                                             0.4 ± 0.1 7.0 ± 1.1                                        Latex-injected                                                                             0.8 ± 0.09                                                                              0.6 ± 0.05                                       Rabbit                                                                        ______________________________________                                    

Table 5 clearly shows that priming which results from prior treatmentwith non-phagocytosable particles was insignificant. Contrasting Table 5with the results above, clearly the size of the particle plays animportant role in priming macrophages for enhanced killing potential.

Failure of Zymosan to Prime Normal AM In Vitro

To determine whether zymosan particles can prime normal resident AM invitro, freshly harvested AM from normal rabbits were incubated with 5 mgzymosan/ml of AM suspension in RPMI 1640 medium for 18 hours at 37° C.in 5% CO₂. AM incubated without zymosan served as controls. Afterincubation, the cells were washed and assayed for CL responses using PMAas the eliciting agent. Table 6 shows that zymosan particles did notprime normal AM in vitro for enhanced oxidative responses as they didwhen injected i.v. into rabbits.

                  TABLE 6                                                         ______________________________________                                        Treatments    Chemiluminescence (CPM* 10.sup.-4)                              of AM         Resting      Peak                                               ______________________________________                                        AM incubated alone                                                                           0.1 ± 0.02                                                                             2.5 ± 1.1                                       AM incubated with                                                                           0.06 ± 0.01                                                                             0.4 ± 0.06                                      zymosan                                                                       ______________________________________                                    

Table 6 shows that incubating AM for 18 hours with zymosan particlesactually resulted in a reduced level of the oxidative responses.Incubating normal AM for 18 h resulted in an enhanced PMA-elicited CLresponses (25,000 CPM) compared to the level of CL responses generatedby freshly harvested AM (3,000 to 5,000 CPM). This phenomenon wasreferred to as "spontaneous priming" by Hayakawa et al., J. Leuk. Biol.,45:231-238 (1989), which is noted above.

In Vitro Priming of Normal AM with Bronchoalveolar Lavage Fluids (BAL)Produced from Zymosan Injected Rabbit

FIG. 1 shows that when freshly harvested AM from normal rabbits wereincubated for three hours with BAL procured from rabbits injected withzymosan particles three days prior to harvesting cells, it primed normalAM for more than as 2-fold increase in PMA-elicited CL responsescompared to untreated AM. In FIG. 1, rabbits were injected i.v. with 20mg of zymosan in 2 ml of saline three days prior to extracting BAL. AMharvested from control rabbits were incubated for three hours at 37° C.with BAL preparations (50%) and were subsequently assayed for oxidativeresponses with PMA as the eliciting agent. It is noted that the level ofoxidative burst by AM primed in vitro with BAL was much lower comparedto in vivo priming with particle injection (see Table 1 above).

FIG. 2 shows that when AM from normal rabbits were incubated for 18 hwith various concentrations of BAL fluids procured from zymosan injectedrabbits, BAL fluids primed normal AM for as high as a 15-fold increasein latex elicited CL responses compared to that observed with untreatedAM. In FIG. 2, BAL was extracted from rabbits two days after i.v.injection with 20 mg of zymosan. AM harvested from control rabbits wereincubated with various concentrations of BAL for 18 hours at 37° C. andsubsequently assayed for CL responses with latex as the eliciting agent.FIG. 2 shows that incubation of normal AM with lavage fluids procuredfrom normal rabbits did not prime normal AM.

FIGS. 1 and 2 indicate that BAL fluids of zymosan-injected rabbitscontain a macrophage priming factor (e.g., a cytokine) capable ofpriming macrophages that have not been exposed to particles. Only theBAL fluids of zymosan-injected rabbits contained a macrophage primingfactor(s) that can prime normal AM in vitro. It is anticipated that themacrophage priming factor can be isolated and administered to patientsinstead of the phagocytosable particles to achieve the short durationpriming effect shown in the data above. Hence, patients would beprovided (e.g., by injection (intravenous, intratracheal,intraperitoneal, intramuscular, subcutaneous etc.), aerosol, or othermeans such as suppository, oral or nasal delivery, etc.) with apreparation containing the macrophage priming factor so that themacrophages in that patient could be primed for enhanced killingpotential a certain number of days after such administration. It wasobserved that the macrophage priming factor present in BAL is relativelyunstable and that about 50% of its activity is lost on storage at -60°C. over a 15-day period.

3. Applications

The important points discovered in the above experiments were: (1) theinjection of adult rabbits with particulate preparations (zymosan,latex, or HK-BCG) of phagocytosable size prime AM in vivo in 1 to 4 daysto give a very large oxidative burst when elicited in vitro with PMA,Op-zym or latex; (2) AM cannot be primed in vitro with the particulatepreparations used; (3) AM are not primed in vivo by injecting largenon-phagocytosable particle preparations; and (4) normal AM are primedin vitro with BAL procured from zymosan injected rabbits.

It is of particular interest that the magnitude of the elicitedoxidative burst observed in the experiments of AM primed by i.v.injection of particulate preparations was equal to the maximal primingachieved three weeks after immunization with heat-killed BCG in oil(See, Chida et al., Infect. Immun.,55:1476-1483 (1987), Giridhar et al.,J. Leuk. Biol., 49:442-448 (1991), and Hayakawa et al., J. Leuk. Biol.,45:231-238 (1989)). This level of response is indeed impressive becauseit represents more than a 100-fold increase in the capacity of AM togenerate oxygen radicals as compared to resident AM from normal animalswhen elicited in vitro. This response is markedly different fromclassical T cell-mediated priming of macrophages in that thepost-injection interval is only two to three days before maximal primingis observed.

The fact that AM cannot be primed in vitro with the particulatepreparations used is of particular interest. This suggests that a secondcell type may be involved in the priming of AM. A requirement of aparticle of phagocytosable size is also notable. We were unable toinduce any detectable priming of AM with non-phagocytosable latexparticles. The requirement that particles must be of phagocytosable sizesuggests that phagocytosis of the injected particulates triggered theproduction of some macrophage-derived cytokine that activated asecondary cell type, such as a lymphoid, which is ultimately responsiblefor production of a priming factor(s). In this regard, eitherinterleukin-1 or tumor necrosis factor could be candidates foractivating the cell that ultimately synthesizes a priming factor(s). Theobservation that the lavage fluid obtained from particulate-injectedrabbits primed normal AM in vitro (FIGS. 1 and 2) indicates a primingfactor(s) accumulates in the lungs of injected rabbits.

The priming mechanism discovered, which has a rapid and short term,represents a non-specific form of a cell-mediated defense system. Thepotential of more than a 100-fold increase in the oxidative responses oflung macrophages and the associated killing capacity will have a highlybeneficial effect in controlling lung infections, as well as otherinfections, under circumstances in which classical cell-mediatedimmunity does not have time to develop.

It is anticipated that in situations where patients who are about toundergo a planned surgery, or where soldiers are about to undergo aplanned invasion or encounter biological weaponry, or in any othersituation contagion will be encountered, a person could be provided witheither preparations of phagocytosable particles or preparationsincluding a macrophage priming factor one to four days, and morepreferably two to three days, prior to the event.

The administration of the particles could be by injection, inhalation ofan aerosolized dose, or by other suitable means.

In view of the particle preparations which were effective in priming themacrophages for significantly enhanced activity in rabbits, a suitabledose range for administration to human beings would be between 0.5 and 2mg per kg of body weight. These estimations are based on the rabbit datathat would produce about 75% of the maximal response. It is expectedthat higher dosages may be possible. Providing enough particles for themaximal response would be a goal. It is critical that the particulatesused for priming of macrophages be phagocytosable (e.g., between 0.3 and5 μm in diameter). In view of the results above, almost any type ofparticle would be suitable for quickly priming macrophages to anenhanced killing potential. It is preferred that the particles bebiodegradable. For example, suitable biodegradable particles wouldinclude: biodegradable microspheres that are compounds of L-lacticacid/glycolic acid homo- and co-polymers (see, Tabata et al., J. Biomed.Mat. Res. 22:837-858 (1988)); gelatin particles (cross-linked);degradable starch complexes; biodegradable hydrogel such aspoly(2-hydroxy-ethyl-L-glutamine) (PHEG) (see, Merchant et al., J.Biomed Mat. Res. 17:301-325 (1983)); hydroxybutyrate-hydroxyvaleratecopolymers (see, Yasin et al., Biomaterials 13:9-16 (1992));concanavalin A; colloidal particles of organic origin; degradablepolyesters including block copolymer poly(ethylenesuccinate)-b-poly(ethylene glycol) (PES/PEG) (see, Albertsson et al.,Acta Polymerica 30:95-104 (1988)); chitin; and cellulose. Ifbiodegradable particles are used, they should remain relatively intactin the body for the 1-4 days required optimum short-term priming ofmacrophages.

A major point of interest is that the interval required for maximalparticle-induced priming coincides with the 3- to 4-day intervalcommonly observed as the period between bacterial contamination,colonization, and apparent infection following surgery. If priming ofmacrophages could be achieved during this interval, the risk ofinfection may be greatly reduced. Hence, patients which have beenexposed to contagion (viruses or bacteria) could be provided with asuspension phagocytosable particles so that in the short term, thepatient's macrophages could be primed for enhanced killing potentialwithin a short time period (1-4 days).

There are two lines of rationale that support the proposition that amacrophage priming system might be helpful to cancer patients. First, ithas been established that some tumors are destroyed by activatedmacrophages, especially sarcomas. Second, some tumors of lymphoid cellorigin cause a marked immunodepression which can result in severeopportunistic infections. Hence, a system like that which has beendisclosed which primes macrophages to a high level of anti-tumoractivity as well as anti-microbial activity could have beneficialeffects in such patients. In addition, the macrophage priming capabilityof the inventive system will be useful in patients suffering from theacquired immune difficiency syndrome (AIDS). AIDS and cancer patientshave increased susceptibility to secondary infections and theirmacrophage system is usually preserved until very late stages in theirdisease. Therefore, this macrophage augmentation effect should beextremely useful in preventing and treating secondary and opportunisticinfections. As the effects last one week and are expected to benon-toxic to tissue cells, the administration may be repeated at monthlyintervals for both cancer and AIDS patients.

Particle preparations may be administered by intravenous orintraperitoneal injection. The preparations may be prepared in saline aswell as conventional buffers to render the injectable particlesuspensions isotonic.

Aerosol delivery of the particles might also be used (e.g. via anebulizer or metered dose inhaler). A chief advantage of aerosolizationof the particles would be the non-invasive delivery procedure. If theparticles are formulated into a metered dose inhaler (MDI) for aerosoldelivery to the lungs, it will need to be dispersed in a propellant andpackaged in a canister under pressure. The propellant could be any or acombination of the commonly used freons or CFCs, such as CCl₃ F (Freon11 or CFC-11), CCl₂ F₂ (Freon 12 or CFC-12), and CClF₂ -CClF₂ (Freon 114or CFC-114). However, there has recently been much emphasis on usingmore ozone friendly propellants such as 1,1,1,2-tetrafluoroethane(HFC-134a) and propellant 227, hydrocarbons (propane, butane, isobutane,etc.), fluorocarbons (perfluoropentane), dimethyl ether, or the like, inMDI applications and any of these gases or combinations thereof could beused. As with almost all MDI applications, the propellant typicallyconstitutes over 90% by weight of the composition mixture. Surfactantssuch as oleic acid, lecithin, sorbitan trioleate, and the like, mightalso be included for lubricating the metering valve and aiding indispersing the particles within the mixture.

As discussed above, alternative preparations for short term priming ofmacrophages would include the macrophage priming factor released bycells in response to encountering the phagocytosable particles. As shownin FIGS. 1 and 2, a macrophage priming factor exists in BAL fluids fromanimals exposed to phagocytosable particles in vivo two or three daysprior to harvesting AM. This macrophage priming factor could be obtainedfrom fluids in animals pretreated with phagocytosable particles or byrecombinant or other suitable techniques. Preparations containing themacrophage priming factor, similar to the particle preparationsdescribed above, could be prepared for delivery by aerosol, i.v. or i.por i.t. injection, or by other suitable means. The macrophage primingfactor would be dissolved or dispersed in a pharmaceutically acceptablecarrier fluid or gas or binder or elixir which would facilitateproviding the macrophage priming factor to the patient.

While the invention has been described in terms of its preferredembodiments, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims.

Having thus described our invention, what we claim as new and desire tosecure by Letters Patent is as follows:
 1. A method for primingmacrophages in vivo in patients in need of prophylactic treatment forbacterial and viral infections, comprising the steps of:day when aproviding patient enhanced macrophage activity by administering to saidpatient an effective dose of phagocytosable particles selected from thegroup consisting of biodegradable particles, opsonized zymosanparticles, PMMA latex, polystyrene, heat killed-BCG, and heat killedStaphylococcus epidermis one to four days prior to said day when saidpatient requires enhanced macrophage activity, whereby saidphagocytosable particles prime macrophages in said patient for enhancedmacrophage activity.
 2. A method as recited in claim 1 wherein said stepof administering is performed by injection.
 3. A method as recited inclaim 1 wherein said step of administering is performed by inhalation.4. A method as recited in claim 1 wherein said phagocytosable particlesare biodegradable in a patient's body more, than four days afteradministration.
 5. A method for priming macrophages in vivo in patientsin need of enhanced bactericidal and virucidal activity, comprising thesteps of:administering to a patient that has been infected with abacterial or viral agent and would benefit from enhanced macrophageactivity an effective dose of phagocytosable particles selected from thegroup consisting of biodegradable particles, opsonized zymosan, PMMAlatex, polystyrene, heat killed-BCG, and heat killed Staphylococcusepidermis, whereby said phagocytosable particles prime macrophages insaid patient for enhanced macrophage activity one to four days afteradministration.
 6. A method as recited in claim 5 wherein said step ofadministering is performed by injection.
 7. A method as recited in claim5 wherein said step of administering is performed by inhalation.
 8. Amethod as recited in claim 5 wherein said phagocytosable particles arebiodegradable in a patient's body more than four days afteradministration.